Activation of p44/42 mitogen activated protein kinases in thrombin-induced brain tolerance

BACKGROUND: Our recent studies have shown that prior intracerebral injection of a low dose of thrombin attenuates the brain edema formation that results from either an intracerebral hematoma, an intracerebral injection of a large dose of thrombin or cerebral ischemia. The aim of the current study is to investigate whether thrombin-induced tolerance (thrombin preconditioning; TPC) is associated with activation of p44/42 mitogen activated protein (MAP) kinases. METHODS: This study contained three parts. In the first, rats received an intracerebral infusion of either saline or one unit thrombin (the TPC dose) into the right caudate nucleus. After 1, 3 and 7 days, the rats will be killed and brains used to detect p44/42 MAP kinases activation using Western blot analysis and immunohistochemistry. In the second and third parts, rats received intracerebral infusions of either vehicle, one unit thrombin (TPC) or one unit thrombin and 5 nmol PD 098059. These rats were either killed to detect kinases activation after 24 h or received a second intracerebral infusion of five-unit thrombin 7 days later with brain edema being assessed after a further 24 h. RESULTS: Western blot analysis demonstrated that p44/42 MAP kinases were activated in the ipsilateral basal ganglia after the intracerebral infusion of thrombin one unit. Cells immunoreactive for activated p44/42 MAP kinases were found in the ipsilateral basal ganglia and ipsilateral cortex. PD 098059, a MAP kinase kinase inhibitor, abolished thrombin-induced activation of p44/42 MAP kinases. TPC suppressed thrombin-induced brain edema while PD 098059 blocked this protective effect. The water contents in the ipsilateral basal ganglia 24 h after infusion of thrombin five units were 82.6+/-0.8%, 79.2+/-0.4% and 81.8+/-1.9% in the control, TPC alone and TPC plus PD 098059 groups, respectively. CONCLUSION: Thrombin can activate p44/42 MAP kinases within the brain and the protective effects of thrombin preconditioning on brain edema formation are related to this activation.     

吡那地尔预处理对失血性休克大鼠脏器热休克蛋白70和细胞型磷脂酶A2表达的影响

目的 探讨吡那地尔（Pinacidil)预处理（preconditioning,PC)对失血性休克大鼠保护作用的机制。方法 （1）大鼠90只,随机分为正常组（N组,10只）、对照组（C组,40只）和预处理组（PC组,40只）,PC组大鼠用Pinacidil进行预处理,24h后将对照组和预处理组大鼠复制成失血性休克模型,用Westem blot方法观察不同时相点大鼠心肌组织细胞型磷酯酶A2（cPLA2）的表达变化。（2）大鼠60只,随机分为对照组（C组,30只）和预处理组（PC组,30只）,PC组大鼠用Pinacidil进行方法观察PC对大鼠心肌和肝脏组织热热休克蛋白70（hsp70)表达的影响。结果 （1）PC组心机组织cPLA2的表达较弱,而C组表达很强;（2）C组hsp70表达很弱或检测不到,PC组心肌和肝脏hsp70表达很强,24h达到高峰,48h和72h逐渐减弱。结论 Pinacidil预处理可能通过诱导hsp70过度表达和抑制PLA2的表达保护“休克细胞”,从而保护失血性休克大鼠的心肌和肝脏组织。     

缺血预处理对肝脏抗氧化能力的影响及机制

目的 在大鼠肝脏原位缺血再灌注(I/R)模型上观察缺血预处理(IP)对肝脏抗氧化能力的影响及其机制。方法 在大鼠肝脏原位缺血再灌注模型上,心包穿刺抽血,切取肝组织,分别进行血清ALT、AST检测,肝组织MDA、SOD、Cat、GSH-PX测定。结果 发现预处理组与缺血再灌注组相比血清中ALT、AST水平及肝组织匀浆中MDA的含量降低,而肝组织中SOD、Cat及GSH-PX的活性均不同程度增高,多数检测指标各预处理组之间均无明显差异。结论 实验表明缺血预处理能增强缺血再灌注肝脏的抗氧化能力,增加预处理的次数并不能增加预处理的效果。腺苷在缺血预处理过程中起着重要作用。     

Mitochondrial adaptations within chronically ischemic swine myocardium

Experimental evidence has emerged that myocardial ischemic preconditioning can prime the mitochondria into a "stress-resistant state", so that cell death is reduced following prolonged severe ischemia and reperfusion. Using a swine model of chronically ischemic myocardium, we tested the hypothesis that mitochondria within the ischemic territory have also acquired a protective phenotype. Eleven swine underwent a left thoracotomy with placement of an external constrictor around the proximal left anterior descending (LAD) artery. By 10 weeks, a severe stenosis of the LAD artery was documented by quantitative coronary angiography (92 +/- 2%). Animals were sacrificed and myocardium was extracted from the LAD and remote regions. Mitochondria were isolated from subendocardium and subepicardium from LAD and remote regions and state 2 (substrate alone) and state 3 (+ADP) respiration were assessed with a Clark electrode. Within the LAD subendocardium, the respiratory control index was 2.68 +/- 0.17 and was lower than the remote subendocardium (3.64 +/- 0.08; P < 0.05). When exposed to 20 min anoxia with reoxygenation, the LAD region demonstrated a more preserved state 3 respiration compared with the remote region (99 +/- 14 versus 65 +/- 9 nmol O2/mg, respectively; P < 0.05). In parallel mitochondrial experiments, chemiluminescence was detected with the probe coelenterazine and superoxide generation in the LAD region in the presence of antimycin A was 574 +/- 108 RLU/30 s/microg and was nearly 50% lower than the remote region (979 +/- 175 RLU/30 s/microg; P < 0.05). Within the mitochondria, the expression of uncoupling protein (UCP) 2 by western gels was 20% higher in the LAD region compared with the remote region (P < 0.05) with no differences noted in UCP-3. In this swine model of chronic myocardial ischemia, isolated mitochondria from the ischemic tissue demonstrate preserved state 3 respiration following anoxia/reoxygenation, consistent with a stress-resistant state. This state is characterized by a mild degree of uncoupling under basal conditions and decreased superoxide generation. Uncoupling protein 2 expression is enhanced in the mitochondria, providing a potential mechanism for these favorable mitochondrial adaptations.     

Short-term ischemia usually used for ischemic preconditioning down-regulates central cannabinoid receptors in the gerbil hippocampus

Transient forebrain ischemia of 5-min duration causes delayed neuronal death (DND) of vulnerable CA1 neurons in the gerbil hippocampus, which can be prevented by preconditioning with a short ischemic stimulus of 2.5-min duration. While a key role of excitatory glutamate receptors for both phenomena has been widely accepted, little is known about the postischemic regulation of central cannabinoid (CB1) receptors. The present study was designed to test whether ischemic preconditioning is associated with specific alterations of protein expression and/or ligand binding of these receptors compared to ischemia severe enough to induce DND. Gerbils were subjected to either a 5-min ischemic period resulting in DND of CA1 neurons, or a 2.5-min period of ischemia usually used for preconditioning. Postischemic hippocampal CB1 receptor protein expression was investigated immunohistochemically, while postischemic ligand binding of [³H]CP 55940 to CB1 receptors was analyzed by quantitative receptor autoradiography in both experimental groups after 24, 48, and 96 h (n=4–5 per time point), respectively, and compared to sham-treated gerbils (n=10). Short-term ischemia of 2.5-min duration caused a transient reduction of hippocampal CB1 receptor protein expression, while receptor binding density was permanently decreased. In contrast, 5-min ischemia did not alter protein expression or ligand binding up to 48 h. Based on these data, postischemic down-regulation of hippocampal CB1 receptors, specifically seen after short-term ischemia usually used for preconditioning, may participate in the mechanisms of endogenous postischemic neuroprotection.The first two authors contributed equally     

心肌缺血预适应和后适应影响因素的研究

急性心肌缺血是危害人类生命的原因之一,尽快恢复血液供应是急性心肌缺血的根本治疗手段,但突然恢复血液灌注会出现再灌注损伤。大量的研究证实,心肌缺血预适应和后适应可以减轻再灌注损伤,但具体机制尚未阐明。研究过程中,动物种属、刺激方案、高血糖、高血脂等很多因素可能会干扰心肌缺血预适应和后适应的研究结果,撰文对这些影响因素予以综述。     

缺血预处理对犬脊髓损伤及热休克蛋白70表达影响的研究

目的评价缺血预处理对犬脊髓损伤及热休克蛋白70表达的影响。方法41条杂种犬随机分成假手术组6只、预处理组21只、对照组14只。预处理组主动脉阻断6min后开放6min,反复2次,之后阻断35min;对照组主动脉阻断35min。术后进行神经功能评分,检测脊髓组织中热休克蛋白70表达。结果在再灌注后6h、24h预处理组热休克蛋白70于胞质和胞核均有表达,且强于对照组;而且神经功能评分预处理组高于对照组。在再灌注后7d预处理组神经功能评分无明显改变,且仍见热休克蛋白70表达。结论缺血预处理可以增加脊髓的缺血耐受;热休克蛋白70在胞质和胞核中表达可能在缺血耐受中起到一定的作用。     

针灸预处理对缺血再灌注大鼠肌细胞凋亡及C—fos基因调控

目的:探求针灸预处理对心肌缺血再灌注损伤大鼠细胞凋亡及C-fosmRNA的影响。方法:采用针灸预处理组间,针灸预处理与缺血预处理比较,利用大鼠心肌缺血再灌注模型观察,分正常对照组、假手术组、缺血再灌注组、缺血预处理组、手针预处理日一次组、电针预处理日一次组、艾灸预处理日一次组、手针预处理日两次组、电针预处理日两次组、艾灸预处理日两次组、结果:表明针灸预处理使C-fosmRNA增多,使细胞凋亡减少,且针灸预处理日两次组比日一次组和缺血预处理更有效。结论:针灸预处理日两次组更有效预防心肌缺血再灌注损伤大鼠细胞凋亡,进一步证明了中医针灸治未病理论科学价值。     

左-卡尼汀预处理对缺血再灌注大鼠心肌的保护作用

目的 探讨左一卡尼汀（L-CN）预处理对大鼠心肌缺血再灌注损伤（MIRI）的心肌保护作用及其可能的机制。方法 36只Wistar大鼠随机分为3组，每组n=12。左一卡尼汀治疗组（L—CN）：术前给予L-CN200mg·kg^-1·d^-1腹腔注射给药5天；缺血一再灌注组（IR）：术前给予等量生理盐水腹腔注射5天；假手术组（P）：术前处理同IR组。L-CN和IR两组大鼠在心电监护下结扎左前降支（LAD）30min后松开结扎线，恢复心肌血流灌注3h后处死动物，P组除LAD仅穿线不结扎外，所有操作同前两组。（1）三组均于术前和处死动物前从颈动脉抽血观察血浆肌酸激酶、MB同工酶（CK-MB）、丙二醛（MDA）含量及超氧化物歧化酶（SOD）活性。（2）实验结束处死大鼠前留取心肌标本，光镜下观察心肌病理学改变，TUNEL法测定心肌细胞的凋亡指数。结果（1）动物处死前，IR组MDA、CK-MB高于L-CN组（P〈0．01），而SOD低于L-CN组（P〈0．01）。（2）IR组心肌细胞凋亡指数明显高于L-CN组（P〈0．05）。（3）组织形态学观察（HE）：IR组心肌纤维水肿、大部分断裂；L-CN组心肌纤维扭曲，但结构基本完整。结论 L-CN预处理对大鼠MIRI具有一定的保护作用，其机制可能是通过抑制心肌脂质过氧化、减少心肌细胞凋亡。     

Unraveling the mysteries of classical preconditioning

Nearly two decades have passed since the first report about ischemic preconditioning. Although we do not yet know unequivocally what the final effector is, we have learned a lot about the signal transduction pathways that result in protection, and have some good prospects for the final step that results in survival or necrosis of the ischemic myocardium. Many investigators have contributed to our current knowledge. We were heartened to learn that four of our JMCC publications are included in the 20 top-cited papers in the journal's history. It is gratifying that our prior publications have generated some interest and stimulated important debate as documented by the high number of citations by scientists in the bibliographies of their own papers. In this document, we have been asked to reflect on those four papers and comment on where they have led us.